Current human rabies virus (RV) vaccines are effective if administered in an appropriate and timely manner and therefore, RV infection is a vaccine-preventable disease. However, immunization protocols are complex, requiring multiple doses over a period of weeks and in some cases months. High costs and the lack of compliance associated with current vaccines, and the re-emergence and emergence of rabies and rabies- related viruses, helps to keep rabies a global health threat. The World Health Organization estimates RV kills over 55,000 people per year and over 15 million people receive post-exposure prophylaxis (PEP) after exposure to potentially infected animals. Rabies is ranked seventh in important infectious diseases since it often occurs in children. In addition, current vaccines are based on the inactivation of live RV, however, live RV was recently discovered in a production lot of vaccine, resulting in an international vaccine recall and shortage indicating intrinsically safe human RV vaccines are needed. Taken together, the development of novel pre- and post-exposure vaccines is necessary to combat this global health issue. Our overall goal is to develop new human RV vaccines that are safe, inexpensive and effective as pre- and post-exposure vaccines for both industrialized and developing countries. Since pre-exposure vaccination is reserved only for those at-risk populations, such as laboratory workers and veterinarians, PEP is the worldwide standard for human rabies prevention. We hypothesize that a matrix (M) gene-deleted RV, which renders the virus replication-deficient, will make excellent an RV PEP. Among other attributes, M-gene deleted RV vaccines elicit immune responses similar to that from live RV vaccines, which are more potent and different from inactivated vaccines. Importantly, these replication-deficient viruses are also very safe even in T- and B- cell immune-deficient mice and in non-human primates. The M gene-deleted RV emerged as our most promising vaccine vector identified during Phase I studies in conjunction with our other preliminary immunogenicity and protection data in mice non-human primates. We believe M-gene deleted RVs will benefit PEP vaccination reducing the number of inoculations from the current standard regimen of five doses of active and one dose of passive immunization to a one- or two-dose immunization protocol. Three Aims are proposed to achieve our overall goal. Aim I is directed towards preparing a research master seed of the M gene-deleted RV using protocols developed during Phase I to recover and propagate M gene-deleted RVs on Vero cells (a pharmaceutically acceptable cell substrate). Aim II is directed towards assessing the safety and toxicity of the M gene-deleted RV by studying its biodistribution, histopathology, genetic stability, thermal stability and neurovirulence in mice. Aim III is to further evaluate immunogenicity our vaccine vector, and to define or establish a new acceptable potency assay for the replication-deficient virus vaccine by correlating focus forming units (ffu) to the well-defined NIH potency test of RV vaccine-induced immunity and protection. In summary, this Phase II study should finalize pre-clinical testing of the M gene-deleted RV and support the development of a one- to two-dose RV vaccine. Achieving these Aims will bring us closer to saving lives and reducing the cost of human RV prevention in both industrialized and developing countries. PUBLIC HEALTH RELEVANCE: The goal of this application is to develop safe and effective alternatives to the current human rabies post- exposure prophylaxis. The development of treatment that relies on only one to two doses of vaccine instead of five or six inoculations will greatly enhance the effectiveness of rabies virus prevention, save lives and reduced costs in developing and developed countries.